Engineering Disulfide Cross‐Links in RNA Via Air Oxidation

Emily J. Maglott1, Gary D. Glick1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.4
DOI:  10.1002/0471142700.nc0504s00
Online Posting Date:  May, 2001
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Abstract

This unit presents protocols for the synthesis of alkylthiol‐modified ribonucleosides, their incorporation into synthetic RNA, and the formation of intramolecular disulfide bonds in RNA by air oxidation. The disulfide bonds can be formed in quantitative yields between thiols positioned in close proximity by virtue of either the secondary or tertiary structure of the RNA. Disulfide cross‐links are useful tools to probe solution structures of RNA, to monitor dynamic motion, to stabilize folded RNAs, and to study the process of tertiary structure folding.

     
 
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Table of Contents

  • Basic Protocol 1: Formation of Intramolecular Disulfide Cross‐Links in RNA
  • Support Protocol 1: Synthesis of RNA Containing tert‐Butyldisulfide‐Modified Nucleosides by Solid‐Phase Methods
  • Support Protocol 2: Purification of Synthetic RNA Containing tert‐Butyldisulfide Protected Thiol‐Modified Nucleosides
  • Support Protocol 3: Quantification of Thiols in RNA Using 7‐Diethylamino‐3‐(4′‐Maleimidylphenyl)‐4‐Methylcoumarin
  • Support Protocol 4: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐Butyldimethylsilyl)‐N3‐(Ethyl)Uridine‐3′‐O‐(N,N‐Diisopropyl‐β‐Cyanoethylphosphoramidite) tert‐Butyl Disulfide (S.5)
  • Support Protocol 5: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(tert‐Butyl‐Dimethylsilyl)‐2′‐O‐Succinyl‐CPG N3‐(Ethyl)Uridine tert‐Butyl Disulfide Controlled‐Pore Glass Support
  • Support Protocol 6: Synthesis of 3′,5′‐O‐(Tetraisopropyldisiloxane‐1,3‐Diyl)‐2′‐O‐ Allyl‐O4‐(2‐Nitrophenyl) Uridine (S.10) Intermediate
  • Support Protocol 7: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐N4 ‐(Benzoyl)‐2′‐O‐ (Ethyl)Cytidine‐3′O‐(N,N‐Diisopropyl‐β‐Cyanoethylphosphoramidite) tert‐Butyl Disulfide (S.20)
  • Support Protocol 8: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(Ethyl)Uridine‐3′‐O‐(N,N‐Diisopropyl‐β‐Cyanoethylphosphoramidite) tert‐Butyl Disulfide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Formation of Intramolecular Disulfide Cross‐Links in RNA

  Materials
  • tert‐Butyl‐disulfide‐modified RNA (see protocol 2)
  • 100 mM sodium phosphate buffer, pH 8.3 (see recipe)
  • Dithiothreitol (DTT)
  • Sodium phosphate buffer/NaCl, pH 7.0 (see recipe)
  • 5000‐molecular‐weight‐cutoff (MWCO) cellulose ester membrane
  • 200 mM MgCl 2 (see recipe)
  • 0.1 N NaOH
  • TE buffer ( appendix 2A)
  • 1.5 M sodium acetate (NaOAc), pH 5.5
  • Absolute ethanol
  • 10‐well (500 µL) microdialyzer (e.g., Spectrum)
  • 0.2‐µm bottle‐top filter
  • Argon (Ar) tank
  • Peristaltic pump
  • 70°C water bath
  • pH meter with microelectrode
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (e.g., CPMB UNIT and appendix 3B of this manual) or for derivatization and fluorescence spectroscopy for quantitation of thiols (see protocol 4)

Support Protocol 1: Synthesis of RNA Containing tert‐Butyldisulfide‐Modified Nucleosides by Solid‐Phase Methods

  Materials
  • Methylene chloride (CH 2Cl 2) in CaH 2
  • Acetonitrile (CH 3CN) in CaH 2
  • Thiol‐modified nucleoside phosphoramidites (see Support Protocols protocol 54, protocol 87, and protocol 98)
  • Nucleoside phosphoramidites
  • Argon (Ar)
  • Anhydrous ethanol
  • Absolute ethanol, room temperature and –20°C
  • Brine (saturated aqueous NaCl)
  • Nitrogen (N 2) stream
  • Ammonia gas (NH 3)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF)
  • 1.5 M sodium acetate (NaOAc), pH 5.5 (see recipe)
  • Ethyl acetate (EtOAc)
  • Rotoevaporator
  • Automated nucleic acid synthesizer
  • Trap‐Pak molecular sieve bags (Perseptive Biosystems)
  • Rubber septa
  • Cannula
  • CPG column loaded with appropriate nucleoside (purchased, or see protocol 6)
  • Desiccator
  • 1‐ and 2‐dram glass vials (oven dried >8 hr at 180°C)
  • Teflon tape
  • Dry bath, 55°C
  • Speedvac evaporator or equivalent
  • Spatula, RNase‐free
  • 5‐in. (12.5‐cm) glass pipets
  • Rotary shaker

Support Protocol 2: Purification of Synthetic RNA Containing tert‐Butyldisulfide Protected Thiol‐Modified Nucleosides

  Materials
  • Crude synthetic tRNA (see protocol 2)
  • 80% formamide containing 0.05% xylene cyanol (XC) tracking dye
  • 8% denaturing polyacrylamide solution (see recipe)
  • 1× TBE electrophoresis buffer ( appendix 2A)
  • 1× and 4× TAE electrophoresis buffer ( appendix 2A)
  • 1.5 M sodium acetate (NaOAc), pH 5.5
  • Absolute ethanol
  • Power supply
  • Silica‐gel plate
  • UVG‐11 Mineralight lamp (254 nm, 115 V) or equivalent
  • Razor blade, RNase free
  • Hoefer Six‐Pac electroeluter or equivalent
  • Inner elution tubes
  • Porous polyethylene plugs
  • Blotter‐paper discs
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (e.g., see CPMB UNIT and appendix 3B of this manual)

Support Protocol 3: Quantification of Thiols in RNA Using 7‐Diethylamino‐3‐(4′‐Maleimidylphenyl)‐4‐Methylcoumarin

  Materials
  • RNA solution containing free thiols (see protocol 1)
  • 5× CPM buffer (see recipe)
  • 0.4 mM CPM in isopropyl alcohol (see recipe)
  • 1% Triton X‐100
  • Sodium phosphate buffer/NaCl, pH 7.0 (see recipe)
  • Fluorimeter and cuvette

Support Protocol 4: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐Butyldimethylsilyl)‐N3‐(Ethyl)Uridine‐3′‐O‐(N,N‐Diisopropyl‐β‐Cyanoethylphosphoramidite) tert‐Butyl Disulfide (S.5)

  Materials
  • Uridine
  • Distilled acetonitrile (CH 3CN)
  • Distilled triethylamine (Et 3N)
  • N,N‐Dimethylformamide (DMF)
  • Chlorotrimethylsilane (TMSCl)
  • N 2
  • Petroleum ether
  • Diethyl ether (Et 2O)
  • Sodium hydride (NaH)
  • 1‐Tosyl‐2‐benzoylmercaptoethanol (see Glick et al., )
  • 48% (w/v) aqueous HF
  • Methylene chloride (CH 2Cl 2)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Methanol (CH 3OH)
  • Pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • 1‐(tert‐Butylthio)‐1,2‐hydrazine carboxmorpholide (see Wünsch et al., )
  • LiOH⋅H 2O
  • Ethyl acetate (EtOAc)
  • Imidazole
  • tert‐Butyldimethylsilyl chloride (TBDMSCl)
  • Tetrahydrofuran (THF)
  • 2,4,6‐Collidine
  • N‐Methylimidazole
  • Chloro‐N,N‐diisopropylamine‐β‐cyanoethyl phosphine
  • Additional reagents and equipment for flash chromatography ( appendix 3E)

Support Protocol 5: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(tert‐Butyl‐Dimethylsilyl)‐2′‐O‐Succinyl‐CPG N3‐(Ethyl)Uridine tert‐Butyl Disulfide Controlled‐Pore Glass Support

  Materials
  • Compound S.4 (see protocol 5)
  • Pyridine
  • Succinic anhydride
  • 4‐Dimethylaminopyridine (DMAP)
  • Methylene chloride (CH 2Cl 2)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Pentachlorophenol
  • Dicyclohexylcarbodiimide (DCC)
  • Petroleum ether
  • Ethyl acetate (EtOAc)
  • Long‐chain alkyl amino controlled‐pore glass (CPG), 1000‐Å pore size, 100 µmol amino groups/g, 120/200 mesh
  • N,N‐Dimethylformamide (DMF)
  • Triethylamine (Et 3N)
  • Methanol (CH 3OH)
  • Diethyl ether (Et 2O)
  • Acetic anhydride
  • Additional reagents and equipment for flash chromatography ( appendix 3E)

Support Protocol 6: Synthesis of 3′,5′‐O‐(Tetraisopropyldisiloxane‐1,3‐Diyl)‐2′‐O‐ Allyl‐O4‐(2‐Nitrophenyl) Uridine (S.10) Intermediate

  Materials
  • Uridine
  • Pyridine
  • 1,3‐Dichloro‐1,1,3,3‐tetraisopropyldisiloxane
  • Methylene chloride (CH 2Cl 2)
  • Methanol (CH 3OH)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4)
  • Triethylamine (Et 3N)
  • Chlorotrimethylsilane
  • Brine (saturated aqueous NaCl)
  • 2‐Mesitylenesulfonyl chloride
  • 4‐Dimethylaminopyridine (DMAP)
  • 2‐Nitrophenol
  • 1,4‐Diazabicyclo[2.2.2]octane (DABCO)
  • p‐Toluenesulfonic acid monohydrate
  • p‐Dioxane
  • Ethyl acetate (EtOAc)
  • Petroleum ether
  • Tetrahydrofuran (THF)
  • Triphenylphosphine
  • Tris(dibenzylideneacetone) dipalladium(0)
  • Allyl ethyl carbonate
  • Additional reagents and equipment for flash chromatography ( appendix 3E)

Support Protocol 7: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐N4 ‐(Benzoyl)‐2′‐O‐ (Ethyl)Cytidine‐3′O‐(N,N‐Diisopropyl‐β‐Cyanoethylphosphoramidite) tert‐Butyl Disulfide (S.20)

  Materials
  • Compound S.10 (see protocol 7)
  • Tetrahydrofuran (THF)
  • Ammonia (NH 3)
  • N 2
  • Methanol (CH 3OH)
  • Petroleum ether
  • Ethyl acetate (EtOAc)
  • Pyridine
  • N,N‐Dimethylformamide (DMF)
  • Benzoic anhydride
  • Ammonium hydroxide (NH 4OH)
  • N‐Methylmorpholine‐N‐oxide
  • Acetone
  • Osmium tetroxide (OsO 4)
  • Saturated aqueous sodium bisulfite
  • Diethyl ether (Et 2O)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Methylene chloride (CH 2Cl 2)
  • p‐Dioxane
  • Sodium periodate (NaIO 4)
  • Sodium borohydrate (NaBH 4)
  • Methanesulfonyl chloride
  • Triethylamine (Et 3N)
  • Thiobenzoic acid
  • Acetonitrile (CH 3CN)
  • 48% (w/v) aqueous HF
  • 1‐(tert‐Butylthio)‐1,2‐hydrazinedicarboxmorpholide (Wünsch et al., )
  • LiOH⋅H 2O
  • 1 N aqueous sodium citrate
  • 4‐Dimethylaminopyridine (DMAP)
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • N,N‐Diisopropylethylamine
  • Chloro‐N,N‐diisopropylamine‐β‐cyanoethyl phosphine
  • Pressure tube (−78°C)
  • CO 2/isopropyl alcohol (i‐PrOH) bath
  • Additional reagents and equipment for flash chromatography ( appendix 3E)

Support Protocol 8: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(Ethyl)Uridine‐3′‐O‐(N,N‐Diisopropyl‐β‐Cyanoethylphosphoramidite) tert‐Butyl Disulfide

  Materials
  • 2‐Nitrobenzaldoxime
  • 1,1,3,3‐Tetramethylguanidine
  • Acetonitrile (CH 3CN)
  • Compound S.10 (see protocol 7)
  • Ethyl acetate (EtOAc)
  • Sodium sulfate (Na 2SO 4)
  • Methanol (CH 3OH)
  • Methylene chloride (CH 2Cl 2)
  • Acetone
  • N‐Methylmorpholine‐N‐oxide
  • Osmium tetroxide (OsO 4)
  • Saturated aqueous sodium bisulfite
  • Celite
  • 1,4‐Dioxane
  • Sodium periodate (NaIO 4)
  • Diethyl ether (Et 2O)
  • Sodium bicarbonate (NaHCO 3)
  • Sodium borohydride (NaBH 4)
  • Brine (saturated aqueous NaCl)
  • Pyridine
  • Methanesulfonyl chloride
  • N,N‐Dimethylformamide (DMF)
  • Triethylamine (Et 3N)
  • Thiobenzoic acid
  • 48% (w/v) aqueous HF
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • 4‐Dimethylaminopyridine (DMAP)
  • Tetrahydrofuran (THF)
  • LiOH⋅H 2O
  • 1‐(tert‐Butylthio)‐1,2‐hydrazinedicarboxmorpholide
  • 1 N aqueous sodium citrate
  • N,N‐Diisopropylethylamine
  • Chloro‐N,N‐diisopropylamine‐β‐cyanoethyl phosphine
  • Petroleum ether
  • Additional reagents and equipment for flash chromatography ( appendix 3E)
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Literature Cited

Literature Cited
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